4.4. ALS Gene Amplification and Sequencing

To investigate whether mutations in the ALS gene contributed to the metsufuron-methyl tolerance, fresh leaf tissue (100 mg) was collected from plants of the four R. kamoji populations (ten individuals per population) that survived from metsulfuron-methyl treatments in the dose-response experiments. The collected tissue samples were frozen in liquid nitrogen, and total DNA was extracted by using the Plant Genomic DNA Kit (Tiangen Biotech, Beijing, China), following the manufacturer’s instructions. A pair of primers (ALSF: 5′-CTCGCCCGTCATCACCAA-3′ and ALSR: 5′-TCCTGCCATCACCCTCCA-3′) were designed to amplify the ALS gene of ~1600 bp containing the eight known resistance-conferring mutation sites, and the PCR protocols have been described elsewhere [31]. The PCR products were detected with 1% agarose gel and purified using the TIANgel Midi Purification Kit (Tiangen Biotech, Beijing, China). The purified product was sequenced using the ALSF and ALSR primers with the Sanger method by a commercial corporation (Biosune Biotechnology Co., Ltd., Shanghai, China). Alignment and comparison of the sequence data were performed using BioEdit software (Version 7.2.5).